The present invention relates generally to spectrophotometers, to methods for measuring a photosynthetic parameter, and to methods for determining the physiological state of a plant.
Photosynthesis in green plants takes place in two stages, the light reactions, which occur only when plants are illuminated, and the dark reactions, which can occur in the absence or presence of light. In the light reactions chlorophyll and other pigments of the photosynthetic cells absorb light energy and conserve it in chemical form as the two energy-rich products adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide phosphate (NADPH); simultaneously, oxygen is evolved. In the dark reactions, the ATP and NADPH generated in the light reactions are used to reduce carbon dioxide to form glucose and other organic products.
In eukaryotic, photosynthetic cells, both the light and dark reactions take place in the chloroplast. Chloroplasts are surrounded by a continuous outer membrane. An inner membrane system encloses the internal compartment. Inside the latter, and often connected to the inner membrane, are many flattened, membrane-surrounded vesicles or sacs, called thylakoids, which are either single, or arranged in stacks called grana. The thylakoid membranes contain all the photosynthetic pigments of the chloroplast and most of the enzymes required for the primary light-dependent reactions. The fluid in the compartment surrounding the thylakoid vesicles, the stroma, contains most of the enzymes required for the dark reactions (i.e. CO2 fixation).
Light energy is absorbed by photosynthetic pigments located within the thylakoid membranes. The primary light-absorbing pigment is chlorophyll. Photosynthetic cells of higher plants always contain two types of chlorophyll. One is always chlorophyll a, and the second in many species is chlorophyll b. In addition to chlorophylls, the thylakoid membranes contain secondary light-absorbing pigments, together called the accessory pigments, which include various carotenoids. The carotenoid pigments absorb light at wavelengths other than those absorbed by the chlorophylls and thus are supplementary light receptors.
The light-absorbing pigments of thylakoid membranes are arranged in functional sets or clusters called photosystems. The clusters can absorb light over the entire visible spectrum but especially well between 400 to 500 and 600 to 700 nanometers (nm). All the pigment molecules in a photosystem can absorb photons, but a special subset of the molecules, housed in complexes of proteins and cofactors, called the xe2x80x98photochemical reaction centersxe2x80x99 in each cluster ultimately convert the light energy into chemical energy. Other pigment molecules, that function to funnel light into the reaction centers, are housed in light-harvesting complexes. They function to absorb light energy, which they transmit at a very high rate to the reaction center.
There are two different kinds of photosystems: photosystem I (PS I), which is maximally excited by light at longer wavelengths, and has a high ratio of chlorophyll a to chlorophyll b; and photosystem II (PS II), which is maximally activated by light below 680 nm, and contains relatively more chlorophyll b and may also contain chlorophyll c. Photosystem I and Photosystem II are functionally linked by a chain of electron carriers, as shown in FIG. 1.
When light quanta are absorbed by photosystem I, energy-rich electrons are expelled from the reaction center and flow down a chain of electron carriers to NADP+ to reduce it to NADPH. This process leaves a deficit of electrons (an electron hole) in photosystem I. This hole is, in turn, filled by an electron expelled by illumination of photosystem II, which arrives via a connecting chain of electron carriers, including a pool of about 6 plastoquinone molecules per reaction center, the cytochrome b6f complex and plastocyanin. The resulting electron hole in photosystem II is filled by electrons extracted from water. This pattern of electron flow is usually referred to as the xe2x80x9cZ-schemexe2x80x9d. Additionally, absorbed light can be reemitted in the form of fluorescence.
The thylakoid membrane has an asymmetric molecular organization. The electron-transferring molecules in the connecting chain between photosystem II and photosystem I are oriented in the thylakoid membrane in such a way that electron flow results in the net movement of H+ ions across the membrane, from the outside of the thylakoid membrane to the inner compartment. Thus photoinduced electron flow generates an electrochemical gradient of H+ ions across the thylakoid membrane, so that: 1) the inside of the thylakoid vesicles becomes more acid than the outside, storing energy as a difference in pH (known as xcex94pH); and 2) the inside of the thylakoid membrane becomes more positively charged than the outside, storing energy as an electrical field (known as xcex94"psgr"). The sum of energies stored as xcex94pH and xcex94"psgr" drives the synthesis of ATP from ADP and inorganic phosphase, for later use in plant biochemical processes.
Lumen acidification also initiates processes that down-regulate the entire photosynthetic apparatus. The down-regulatory processes reduce the amount of light transferred from the light harvesting pigments to the photosystem II reaction centers, thus protecting the reaction centers from over-exposure to light.
Another type of light-induced electron flow that can take place in chloroplasts is called cyclic electron flow, to differentiate it from the normally unidirectional or noncyclic electron flow of the xe2x80x9cZ-schemexe2x80x9d that proceeds from H2O to NADP+. As shown in FIG. 2, cyclic electron flow involves only photosystem I. It is called cyclic because the electron boosted to the first electron acceptor in photosystem I (an iron-sulfur cluster) by illumination of photosystem I, instead of passing to NADP+, flows back into the electron hole of photosystem I by a shunt or bypass pathway. As shown in FIG. 2, this shunt involves some of the electron carriers of the chain between photosystems I and II, including the pool of plastoquinone molecules, the cytochrome b6f complex and plastocyanin. Thus, illumination of photosystem I can cause electrons to cycle continuously out of the reaction center of photosystem I and back into it. During cyclic electron flow there is no net formation of NADPH, nor is there any oxygen evolution. However, cyclic electron flow is accompanied by proton pumping into the lumen (inside) of the thylakoid vesicle. Thus cyclic electron flow can generate ATP, and this process is referred to as cyclic photophosphorylation. Cyclic electron flow is thought to have two functions: to supply ATP when amply supplied with reducing power in the form of NADPH, and to initiate down-regulation by acification of the thylakoid lumen.
The methods of the invention allow one or more photosynthetic parameters of a plant to be determined by measuring the steady-state turnover rates and resistances to turnover of photosynthetic reactions and protein complexes just after a rapid light-to-dark transition. The relaxation processes that occur just after switching off the light (i.e., the Dark Interval Relaxation Kinetics, abbreviated as DIRK) reflect the processes that occurred in the light, and thus the measurements provide information of the steady-state of photosynthesis. The physiological state of a plant (such as whether the plant is subject to an environmental stress) affects photosynthesis. Thus, the methods of the invention can be used to measure one or more photosynthetic parameters which, in turn, can be used to indicate the presence of one or more plant stresses before they become apparent as lowered crop yields or other visible symptoms.
The present invention also provides kinetic spectrophotometers that can be used, for example, in the methods of the invention to collect spectral data from a plant leaf, and the spectral data can be used to determine a value for a photosynthetic parameter. The kinetic spectrophotometers of the present invention utilize a compound parabolic concentrator (CPC) to direct light generated by a light source onto a sample. The CPC intensifies and diffuses the light from the light source before directing the light onto the sample. The ability of the CPC to intensify light permits the generation of high intensity, short-duration pulses of light, which yield high sensitivity signals. Further, when the kinetic spectrophotometers of the invention are utilized to measure a photosynthetic parameter in a plant leaf, the diffused light emerging from the CPC reduces the effects of light-scattering changes within the leaf.
Kramer and Sacksteder (Kramer D. M. and Sacksteder C. A., Photosynthesis Research 56: 103-112 (1998)) disclose a kinetic spectrophotometer that scatters collimated light from a xenon flashlamp before directing the scattered light onto a sample, such as a plant leaf. The light scattering is mainly achieved by passing the light through a hollow scattering chamber made from a material that efficiently scatters light. A substantial amount of the light entering the scattering chamber is lost, for example by escaping through the entry port leading into the light scattering chamber. Typically, less than fifty percent of the light entering the scattering chamber emerges therefrom and is available to be directed onto the sample. In contrast, in the kinetic spectrophotometers of the present invention, typically more than ninety five percent of light entering the CPC, disposed between the light source and the sample, emerges from the CPC and is available to be directed onto the sample.
In one aspect, the present invention provides kinetic spectrophotometers that each comprise: (a) a light source; and (b) a compound parabolic concentrator disposed to receive light from the light source and configured to (1) intensify and diffuse the light received from the light source, and (2) direct the intensified and diffused light onto a sample.
One embodiment of the kinetic spectrophotometers of the invention comprises: (a) a light source; (b) a compound parabolic concentrator comprising an entry aperture, defining an entry aperture area, and an exit aperture, defining an exit aperture area, wherein the compound parabolic concentrator is (1) disposed to receive light from the light source through the entry aperture; and (2) is configured to intensify and diffuse the light received from the light source, and to direct the intensified and diffused light, through the exit aperture, onto a sample, wherein the entry aperture area is larger than the exit aperture area; (c) a second compound parabolic concentrator comprising an entry aperture, defining an entry aperture area, and an exit aperture, defining an exit aperture area, wherein the second compound parabolic concentrator is (1) disposed to receive, through the entry aperture, light that is transmitted through the sample, or that is emitted by the sample; and (2) that is configured to collimate the received light, and to emit the collimated light through the exit aperture onto a filter, wherein the second compound parabolic concentrator entry aperture area is smaller than the second compound parabolic concentrator exit aperture area; (d) a filter disposed to receive light that is emitted from the second compound parabolic concentrator exit aperture, and that is adapted to block a portion of the light emitted from the second compound parabolic concentrator; and (e) a third compound parabolic concentrator comprising an entry aperture, defining an entry aperture area, and an exit aperture, defining an exit aperture area, wherein the third compound parabolic concentrator is (1) disposed to receive, through the entry aperture, light that passes through the filter; and (2) that is configured to intensify and diffuse the light received from the filter, and to direct the intensified and diffused light onto a light detector, wherein the third compound parabolic concentrator entry aperture area is larger than the third compound parabolic concentrator exit aperture area.
In other aspects, the present invention provides methods for measuring a photosynthetic parameter, the methods comprising the steps of: (a) illuminating a plant leaf until steady-state photosynthesis is achieved; (b) subjecting the illuminated plant leaf to a period of darkness; (c) using a kinetic spectrophotometer of the invention to collect spectral data from the plant leaf treated in accordance with steps (a) and (b); and (d) determining a value for a photosynthetic parameter from the spectral data.
The determined photosynthetic parameter(s) can be used to provide information about the type and amount of photosynthetic activity in a plant leaf, or in a whole plant, or population of plants. Additionally, the determined photosynthetic parameter(s) can be used to ascertain whether the subject plant is experiencing one or more of a variety of environmental and/or physiological stresses, such as temperature stress, drought stress and nutrient stress (including nitrogen stress). Thus, in one aspect, the present invention provides methods for determining the physiological state of a plant comprising: (a) illuminating a plant leaf until steady-state photosynthesis is achieved; (b) subjecting the illuminated plant leaf to a period of darkness; (c) using a kinetic spectrophotometer of the invention to collect spectral data from the plant leaf treated in accordance with steps (a) and (b); (d) determining a value for a photosynthetic parameter from the spectral data; and (e) using the determined value for the photosynthetic parameter to determine the physiological state of the plant.
The kinetic spectrophotometers of the invention are useful, for example, in the practice of the methods of the invention. Additionally, the kinetic spectrophotometers of the invention are useful, for example, to measure the absolute rates of photosynthetic productivity since the initial rates of decay of the electrochromic shift upon a rapid light to dark transition is proportional to the amount of ATP synthesized. The vast majority of ATP is utilized to fix CO2, and so the initial rate of decay of the electrochromic shift should be a good approximation of the rate of CO2 fixation. Further, the kinetic spectrophotometers of the invention are useful to measure any process that requires high sensitivity measurements of absorbance changes in highly scattering samples, or in samples which display large changes in light scattering (e.g., any assay using intact cells or sub-cellular organelles, or in rapid mixing experiments, such as stopped flow experiments).